The promise and potential of gene therapy to cure or alleviate symptoms in a variety of disorders and diseases has stimulated intensive research into new methods for transferring genes into cells. Gene therapy delivered by non-viral plasmid vectors or as linear DNA molecules has been only partially effective in animals and in a clinical setting because of technical difficulties. These difficulties arise from (1) cellular barriers to entry of DNA across the plasma membrane into the cytoplasm, (2) barriers to traversing the cytoplasm intact, and (3) barriers to nuclear entry.
In many targets of gene therapy, it is desirable to transfer and express therapeutic genes only in one cell type within a tissue. Three different approaches have been described for such cell-specific gene expression. First, the delivery of genes to certain cell types is based on the site and physical method of delivery. In other words, by injecting DNA into the big toe, gene transfer will occur mainly in the big toe, not the eye.
The second approach, which has been more widely utilized is to employ cell-specific gene promoters and enhancers to drive gene expression only in desired cell types. In this case, DNA is delivered to all cells within the tissue (or to as many cells as the delivery method allows), and gene expression is limited to those cells in which the promoter is active.
A third way to limit gene expression to certain cell types is to limit nuclear import of the plasmid DNA to certain cell types. Without nuclear import, there is no gene expression and the plasmid or linear gene delivery vehicle is degraded in the cytoplasm. It has been established that the nuclear envelope is one of the major barriers to gene transfer. In non-dividing cells, the nucleus is surrounded by a double membrane envelope that is impermeable to large nucleic acid molecules such as plasmid vectors or linear DNA molecules, which, unlike many proteins, lack discrete signals for nuclear import. Entry of large DNA molecules into the nucleus is very inefficient until the cell enters mitosis and the nuclear membrane temporarily breaks apart.
Specific polynucleotide sequences that facilitate nuclear entry have been identified, however, these sequences have only been successful at targeting entry into the nucleic of certain types of non-dividing cells. For example, a 72 base pair SV40 viral DNA sequence has been shown to facilitate entry of plasmids into the nuclei of a variety of cell types (Dean, Exp Cell Res 253:713, 1999; U.S. Pat. No. 5,827,705).
A sequence present in the smooth muscle gamma actin promoter is a mammalian gene sequence that was recently discovered to possess intrinsic nuclear entry activity. Because the smooth muscle gamma actin promoter DNA binds to a collection of transcription factors only expressed in smooth muscle cells, the DNA sequence mediates nuclear import selectively in smooth muscle cells. In transfection studies, incorporation of this sequence into a plasmid expression vector increases smooth muscle-specific gene transfer and expression; no expression is seen in non-smooth muscle cells. This sequence functions both in cultured cells and for smooth muscle cell selective gene delivery in animals (Vacik et al., Gene Therapy 6:1006, 1999; Dean, U.S. Pat. No. 6,130,207).
The common feature to these nuclear entry or import sequences is that they contain binding sites for transcription factors that in turn harbor protein signals known as nuclear localization signals (NLS) that interact with the cell's machinery for nuclear protein import.
However, to date no such nuclear targeting sequences have been identified that are capable of directing entry into the nuclei of osteoblast lineage cells involved in bone growth and regeneration.